Since interstitial cystitis is likely caused by or exacerbated by defects in the bladder's permeabllity barrier, an understanding of the bladder's barrier function may be critical to understanding the pathogenesis of this disorder. The proposed studies seek to examine, at a fundamental level, the characteristics of this permeability barrier, its molecular basis, and the mechanisms used by bladder epithelial cells to maintain the barrier in the face of the large changes in bladder surface area resulting from filling and emptying the bladder. The first aim is to characterize the permeability properties of intact rabbit bladder epithelial cells to water, protons, and small nonelectrolytes. Rabbit bladders will be stripped of their underlying musculature and mounted in an Ussing chamber where they will undergo measurements of transepithelial voltage, short circuit current, and capacitance. Fluxes of water and small nonelectrolytes such as urea will be measured using isotopes, and proton and NH3 fluxes will be measured using a pH stat. All fluxes will be corrected for unstirred layer effects and normalized to the surface area estimates provided by the capacitance measurements. These results will provide the first direct measurements of the permeability properties of the bladder permeability barrier, and will set the stage for future studies examining the effects of various potential toxins on this tissue. The second aim is to characterize the permeability properties of apical membrane endosomes (AME) which shuttle membrane into and out of the apical surface. Using methods similar to those developed by the PI for studies of toad urinary bladder endosomes, fluorescein derivatives will be entrapped in AME and these vesicles partially purified under conditions in which only AME contain the fluorophore. Using this partially purified preparation, permeabilities to water, small nonelectrolytes, protons, and NH3 will be determined by means of stopped flow fluorimetry. Functional AME will then be purified to homogeneity using a combination of flow cytometry, free flow electrophoresis, and phase separation techniques. These purified AME will be subjected to analysis of lipid composition and extracted lipids from AME will be reconstituted into liposomes to determine if the permeability properties are preserved. These studies will define how lipid composition and bilayer structure determine the permeability properties of the bladder permeability barrier. The third aim is to examine mechanisms by which the trafficking of AME into and out of the apical membrane might be governed. AME will be purified to homogeneity using density shift and flow cytometry approaches and their protein composition determined to identify proteins likely involved in the regulation of AME trafficking. Studies with the flow cytometer will help define whether there are more than one population of AME and will determine the characteristics of each. These studies will provide some initial clues as to mechanisms regulating the insertion and removal of AME from the apical membrane.